A common working hypothesis for the physiological basis of memory is that persistent changes in behavior are mediated by long-term modifications in the strength of synapses (Kandel et al. (1982) Science 218:433-443; Bliss et al. (1993) Nature 361: 31-39). The molecular mechanisms for these changes are complex, involving many signal transduction pathways. Overall, however, these mechanisms are divided into two functionally distinct phases: induction, which initiates the long-term modifications, and maintenance, which sustains the changes (Malinow et al (1988) Nature 335:820-824; Schwartz, J. H. (1993) PNAS 90:8310-8313; Schwartz et al (1987) Ann. Rev. Neurosci. 10:459-476).
Much of the work to examine these signaling pathways has come from the study of the response to high-frequency afferent stimulation of synapses that causes a long-term increase in synaptic transmission, long-term potentiation (LTP) (Bliss et al. (1993), supra.; Bliss et al. (1973) J. Physiol. 232:331-356; Nicoll et al. (1988) 1:97-103). The vast majority of signaling molecules implicated in LTP affect only induction, but not maintenance. The exceptions are agents that inhibit the catalytic domain of protein kinases, specifically protein kinase C (PKC), which are able both to block LTP induction and reverse its maintenance. (Nishizuka, Y (1988) Nature 334:661-665; Schwartz, J. H. (1993) supra; Schwartz et al (1987) supra.
These two phases can be distinguished experimentally by the timing of the application of pharmacological agents that inhibit signal transduction pathways. When agents are applied prior to a tetanic afferent stimulation and prevent the formation of long-lasting changes, they block induction. If they are applied after the tetanus—and reverse the potentiation that has been established—they affect maintenance.
Several principles have been proposed to characterize mechanisms that might maintain long-term changes in synaptic transmission. First, protein kinases, such as PKC, which transiently enhance synaptic transmission when second messengers are activated, can extend their action by becoming constitutively active kinases that are independent of second messengers. (Schwartz et al (1987) supra; Klann et al. (1991) J. Biol. Chem. 266:24253-24256)
Second, long-term forms of synaptic plasticity are thought to depend upon new protein synthesis, although the critical, newly synthesized molecules that cause synaptic potentiation have not been identified. Stanton et al. (1984) J. Neurosci. 4:3080-3088; Frey et al (1988) Brain Res. 452:57-65; Otani et al (1989) Neurosci. 28: 519-526; Abel et al. (1998) Science 279: 338-341. A similar requirement for new protein synthesis has been observed for long-term memory. Davis et al. (1984) Psychol Bull. 96:518-559; Thompson, R. F. Science 233:941-947; Montarola et al. (1986) Science 234:1249-1254.
While usually considered properties of separate mechanisms, it has been determined that one isoform of PKC possesses both of these features: it is persistently increased during LTP as a constitutively active enzyme, and it is generated by new protein synthesis. Sacktor et al. (1993) Proc. Natl. Acad. Sci. (USA) 90:8342-8346. This newly described form of PKC is PKMζ, the independent catalytic domain of the PKCζ isoform, which, lacking PKCζ's autoinhibitory regulatory domain, is autonomously active. Schwartz, J. H. (1993) supra; Sacktor et al. (1993) supra.
PKM is usually thought to be produced by limited proteolysis of PKC, separating the enzyme's regulatory and catalytic domains. This may occur early after a high-frequency tetanus. Recent evidence shows, however, that the long-lasting PKMζ may also be derived from a brain-specific mRNA that encodes only the catalytic domain of ζ. Andrea et al. (1995) Biochem. J. 310:835-843; Powell et al. (1994) Cell Growth Differ. 5:143-149.
PKC is a family of multifunctional protein kinases, first discovered by Nishizuka in 1977. Takai et al. (1977) J. Biol. Chem. 252:7603-7609; Inoue et al. (1977) J. Biol. Chem. 252:7610-7616. PKC consists of two domains separated by a hinge region: an amino-terminal regulatory domain, which contains an autoinhibitory pseudosubstrate sequence and second messenger/lipid binding sites, and a carboxy-terminal catalytic kinase domain. PKC is held in an inactive state in the cytosol by the interaction between the regulatory and catalytic domains. When there is an increase in lipid second messengers (or, for some isoforms, Ca2+), PKC translocates from the cytosolic to membranous (or cytoskeletal) compartments, and a change in its conformation occurs, displacing the regulatory from the catalytic domain, releasing the autoinhibition, and activating the enzyme. The 10 different forms of PKC are divided into 3 groups: conventional (α, βI, βII, γ), novel (or new, δ, ε, η, θ), and atypical (ζ, ι/λ), each of which is activated by a distinct set of second messengers. (PKD or PKCμ is a PKC-related molecule with a catalytic domain closer to CaM-kinase). The conventional PKCs are activated by Ca2+ and diacylglycerol (DAG); the novel by DAG, but not Ca2+ and the atypical by neither DAG or Ca2+, but by alternate lipid-second messengers, including arachidonic acid, phosphoinositide 3-OH kinase products, and ceramide.
A second mechanism for permanently activating PKC, also discovered by Nishizuka, is the cleavage by calpain or their proteases at the hinge region, to permanently separate the regulatory from the catalytic domains. The independently active kinase domain is called PKM. (“M” stands for Mg2+, although this requirement turned out to be for the Mg2+ in Mg2+-ATP). PKM formation results in a persistently active kinase and is not the typical way PKC is activated. It has been found that stable PKM formation occurs endogenously only for a single isoform, ζ, and only in brain. Naik et al. (submitted for publication). Recently, PKMζ has also been reported in a neuronally differentiated cell line. Oehrlein et al. (1998) Eur. J. Cell. Bio. 77:323-337.—Stable PKM forms for the other isoforms have been observed only in pathological conditions: PKMδ in breast cancer tumor cells (Baxter et al. (1992) J. Biol. Chem. 267: 1910-1917) and heart ischemia (Urthaler et al. (1997) Cardiovasc. Res. 35:60-67) and PKMζ in apoptosis (Emoto et al. (1996) Blood 97:1990-1996; Denning et al. (1998) J. Biol Chem. 273:29995-30002).
Protein kinase M zeta (PKMζ) is a form of protein kinase C which has a fundamental role in the formation and maintenance of memory. PKMζ is a critical molecule in the most widely-studied physiological model of memory, long-term potentiation (LTP) of synapses (Sacktor, et al., (1993) supra.; Osten, et al., (1996) J. Neurosci. 16(8):2444-2451; Hrabetova and Sacktor, (1996) J. Neurosci. 16(17):4324-5333).